PHYSIOLOGIC PHOSPHOLIPASE A2 ACTIVATION IN NEUTROPHILS

中性粒细胞中磷脂酶 A2 的生理激活

• 批准号: 2672024
• 负责人: SUE A BAULDRY
• 金额: $ 16.79万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2672024
• 关键词: bacterial toxins; biological signal transduction; calcium indicator; diacylglycerols; enzyme activity; enzyme induction /repression; enzyme mechanism; gas chromatography mass spectrometry; gel electrophoresis; human subject; inflammation; leukocyte activation /transformation; leukotrienes; liquid chromatography; membrane permeability; mitogen activated protein kinase; neutrophil; phosphatidate; phospholipase A2; phospholipase D; platelet activating factor; protein kinase C; radiotracer; second messengers; western blottings

Platelet activating factor (PAF) and leukotriene B4 (LTB4) formation by human polymorphonuclear neutrophils (PMN) is an important aspect of normal PMN function but overproduction of these lipid mediators has been correlated with tissue damage occurring during acute and chronic inflammation. Activation of phospholipase A2 (PLA2) is the initial step in production of LTB4 and PAF and demonstration that PMN contain two calcium-dependent PLA2/s, a "cytosolic" enzyme, cPLA2, and a "secretory" enzyme, sPLA2, has led to intense interest in determining whether one or both are responsible for their formation. This proposal will utilize a permeabilized cell model where cPLA2 is specifically activated in the absence of sPLA2 activity and where both PAF and LTB4 formation occur to determine the actual mechanisms involved in cPLA2 activation. While progress has been made in understanding cPLA2 activation, the question of what mechanism links cell stimulation to cPLA2 activation is still unanswered. In the permeabilized cell model, phospholipase D (PLD) catalyzed production of phosphatidic acid (PA) and diglyceride (DG) are always present when cPLA2 activation occurs, inhibiting PLD activity blocks cPLA2 activation, and adding PA and DG, individually, back to permeabilized cells partially restores cPLA2 activity while a combination of both reconstitutes enzymatic activity. Enhanced PAF and LTB4 formation can be induced in vitro by pretreating PMN with one agonist before stimulation with a second agent by a process called "primed-stimulation". In the permeabilized cell model increased LTB4 and PAF formation occur when cells are primed with the cytokine, tumor necrosis factor, before stimulation. The increased cPLA2 activity is paralleled by enhanced PA and DG formation and blocking PLD activity in primed cells leads to a parallel decrease in LTB4 and PAF production. This proposal will test the hypothesis that PA and DG function as second messengers in both direct activation of cPLA2 and in enhanced cPLA2 activity occurring in primed-stimulation of human PMN. Studies will utilize PMN permeabilized with Staphylococcus aureus alpha-toxin where cPLA2 is maintained in situ and enzyme activation is induced by addition of calcium, guanine nucleotides, and stimulation with a physiologic agonist. In this model, each agent can be added individually or in combination and effects on enzyme activation monitored. Specific Aim 1 will focus on determining the interrelationship between cPLA2 activation and PLD activity, will determine the relative importance of PA and DG in inducing cPLA2 activation and will define the molecular species of PA and DG that effect cPLA2 activity. In Specific Aim 2, requirements for translocation of cPLA2 from cytosol to membrane, for phosphorylation of cPLA2, for activation of the MAP kinase cascade and for activation of protein kinase C will first be determined in the permeabilized PMN, then in cells where PLD activity is blocked and finally in cells where enzyme activity is reconstituted with PA and DG to determine which steps in the signal transduction pathway are effected by PA and DG. This may identify new targets for therapies to combat excessive LTB4 and PAF production occurring during acute and chronic inflammation.

血小板激活因子（PAF）和白三烯B4（LTB4）形成 人类多形核中性粒细胞（PMN）是正常的重要方面 PMN功能，但这些脂质介质的生产过量已经 与急性和慢性期间发生的组织损伤相关 炎。 磷脂酶A2（PLA2）的激活是初始步骤 在LTB4和PAF的生产中，以及PMN包含两个的演示 钙依赖性PLA2/S，一种“胞质”酶，CPLA2和“分泌”酶 酶，Spla2，引起了人们对确定一个还是 两者都负责他们的形成。 该建议将利用 透化细胞模型，其中CPLA2在 没有SplA2活性以及PAF和LTB4形成的情况 确定CPLA2激活中涉及的实际机制。 尽管在理解CPLA2激活方面取得了进展，但 哪种机制将细胞刺激与CPLA2激活联系的问题是 仍未得到答复。 在透化细胞模型中，磷脂酶D（PLD） 催化磷脂酸（PA）和二甘油类（DG）的产生是 CPLA2激活时始终存在，抑制PLD活性 阻止CPLA2激活，并单独添加PA和DG回到 透化细胞部分恢复CPLA2活性，而组合 两种重构酶活性。 可以通过预处理PMN在体外诱导增强的PAF和LTB4形成 用一个激动剂在用过程中用第二代理刺激之前 称为“启动刺激”。 在透化细胞模型中增加 当细胞用细胞因子启动时，LTB4和PAF形成发生， 刺激前肿瘤坏死因子。 CPLA2活性增加 与增强的PA和DG形成并阻止PLD活性并行 引发细胞导致LTB4和PAF产生的平行降低。 该建议将检验PA和DG作为第二的假设 使CPLA2和增强CPLA2中直接激活的使者 在人类PMN的启动刺激中发生的活性。 研究将 利用PMN用金黄色葡萄球菌α-毒素透化，其中 CPLA2原位维持，并通过添加诱导酶激活 钙，鸟嘌呤核苷酸和生理学的刺激 激动剂。 在此模型中，可以单独添加每个代理 对监测酶激活的组合和影响。 具体目标1 将着重于确定CPLA2激活之间的相互关系 和PLD活动将确定PA和DG在 诱导CPLA2激活，并将定义PA的分子种类和 DG影响CPLA2活性。 在特定的目标2中，要求 CPLA2从细胞质转向膜，用于磷酸化 CPLA2，用于激活MAP激酶级联反应和激活 蛋白激酶C将首先在透化PMN中确定 在PLD活性被阻断的细胞中，最后在酶的细胞中 活动与PA和DG重构，以确定哪些步骤 信号转导途径由PA和DG实现。 这可能会识别 与过度LTB4和PAF生产对抗疗法的新靶标 发生在急性和慢性炎症期间。